Protein inducing cell death of helicobacter pylori

ABSTRACT

The present invention identifies the apoptosis-inducing protein of  Helicobacter pylori,  analyzes the structure and provides the amino acid sequence of the protein and the nucleotide sequence encoding the amino acid sequence. Furthermore, the present invention provides inhibiting agents of an apoptosis-inducing activity of the bacterium for the treatment of disease ascribed to the bacterium and a vaccine to prevent the infection of the bacterium.  
     The present invention revealed that the apoptosis-inducing protein of  Helicobacter pylon  is a protein complex composed by association of two kinds of proteins, which comprise a part of the amino acid sequence encoded by γ-glutamyl transferase gene (HP1118). The present invention is said protein and a vaccine comprising said protein.

FIELD OF THE INVENTION

The present invention relates to an apoptosis-inducing protein fromHelicobacter pylori and, more specifically, relates to two novelproteins constituting the apoptosis-inducing protein.

PRIOR ART

Helicobacter pylon infects persistently human stomach and has beenimplicated in the pathogenesis of gastritis and gastric ulcer.Helicobacter pylori infection induces apoptosis in gastric epithelialcells, which plays a significant role in the development of thepathological outcomes. Conventionally, an eradication therapy usingantibiotics has been applied to the Helicobacter pylori infection,however, antibiotics-resistant strains have been emerging and at timesthe eradication therapy by antibiotics is unsuccessful. Consequently, anew methodology of treatment and prevention has been desired.

A method for inhibiting the apoptosis-inducing activity of Helicobacterpylori is a very promising method. By neutralizing theapoptosis-inducing activity of the bacterium, apoptosis in gastricepithelial cells could be inhibited. Therefore, it is highly possible tosuppress the development of gastritis and gastric ulcer.

To date, as the apoptosis-inducing substances produced by the bacterium,Cag A protein, urease, lipopolysaccharide (LPS), and vacuolus toxinshave been studied (WO98/32467 etc.), however, the contribution of thesesubstances in Helicobacter pylori-mediated apoptosis is rather small andthe presence of other unknown factor has been expected for induction ofthe apoptosis (K. Shibayama et al., Infection and Immunity vol. 69, No.5, 3181-3189 (2001)).

On the other hand, the total genome of Helicobacter pylori wasdetermined in 1997 by genome project (Tomb J. F., et al., Nature Vol.388,539-547,1997) and the whole nucleotide sequence has been registeredin a database. However, the nucleotide sequence encoding the proteins,which have an apoptosis-inducing activity, has not been specified.

PROBLEMS TO BE SOLVED BY THE INVENTION

The objective of the present invention is to identify theapoptosis-inducing protein of the bacterium, to analyze the structureand to provide the amino acid sequence of the protein and the nucleotidesequence encoding the amino acid sequence. Further objective of thepresent invention is to provide inhibiting agents of anapoptosis-inducing activity of the bacterium for the treatment of thedisease ascribed to the bacterium and a vaccine to prevent the infectionof the bacterium.

MEANS TO SOLVE THE PROBLEMS

The inventors of the present invention succeeded in purifying and inidentifying the protein with the apoptosis-inducing activity fromHelicobacter pylori and accomplished the present invention. Theapoptosis-inducing protein was found to be composed of two proteins.Analysis of the amino acid sequence of these two proteins and retrievalfrom the database indicated that both these proteins are encoded by theHP1118 gene. The gene is reported to encode γ-glutamyl transferase(Chevalier C., et al., Molecular Microbiology, vol. 31 1359-1372, 1999).However, the apoptosis-inducing activity of the protein has not examinedin their report or in any other reports. Furthermore, γ-glutamyltransferase is a ubiquitous enzyme found throughout bacteria and variousbiological species, and functions to incorporate amino acids intocytoplasm. However, it has not been reported that the enzyme has theapoptosis-inducing activity.

In other words, the present invention demonstrated for the first timethat a protein complex formed by association of two proteins, either ofwhich is constituted by a part of the amino acid sequence encoded byγ-glutamyl transferase gene (HP1118), has an apoptosis-inducing activityof Helicobacter pylori.

Namely, the present invention is an apoptosis-inducing protein ofHelicobacter pylori comprising two kinds of proteins having an aminoacid sequence of SEQ ID NO: 1 or 2, or two kinds of proteins having anamino acid sequence of SEQ ID NO: 1 or 2 wherein one or several aminoacids with respect to the amino acid sequence of SEQ ID NO: 1 or 2is/are deleted, substituted or added, and having an apoptosis-inducingactivity in human gastric adenocarcinoma cell line.

Furthermore, the present invention is a protein comprising either of thefollowing proteins:

(a) a protein having an amino acid sequence of SEQ ID NO: 1.

(b) a protein having an amino acid sequence comprising a deletion,substitution or addition of one or several amino acids with respect tothe amino acid sequence of SEQ ID NO: 1, and having anapoptosis-inducing activity in human gastric adenocarcinoma cell line byassociation with the protein comprising the amino acid sequence of SEQID NO: 2.

Moreover, the present invention is a protein comprising either of thefollowing proteins:

(a) a protein having an amino acid sequence of SEQ ID NO: 2.

(b) a protein having an amino acid sequence comprising a deletion,substitution or addition of one or several amino acids with respect tothe amino acid sequence of SEQ ID NO: 2, and having anapoptosis-inducing activity in human gastric adenocarcinoma cell line byassociation with the protein comprising the amino acid sequence of SEQID NO: 1.

In addition, the present invention is the apoptotic-inducing protein ofHelicobacter pylori comprising said two proteins.

Still furthermore, the present invention is a vaccine comprising one ofsaid proteins, wherein a part of the amino acid sequence of SEQ ID NO: 1and 2 is deleted, substituted or added, and said protein has little orno apoptosis-inducing activity in gastric adenocarcinoma cell line andpossesses a defensive effect against the infection of Helicobacterpylori.

The defensive effect of the infection could be examined by the followingmethods: The animal model for Helicobacter pylori infection has beenestablished using Mongolian gerbil. Candidate protein for vaccination isadministered to Mongolian gerbils by oral administration, transnasaladministration, intramuscular injection or subcutaneous injection toinduce immune response. Subsequently, live Helicobacter pylori areadministered orally and the infection established is examined. Ifdefensive effect against the infection is positive in the animal test,then the effect in human is examined in the same way.

Still moreover, the present invention are respective genes encoding saidtwo kinds of proteins and a method for distinguishing between pathogenicstrains and nonpathogenic strains of Helicobacter pylori, comprising astep of amplifying at least one of the whole or a part of said genes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cytotoxic activity (the absorbance at 450 nm) of eachfraction separated by a cation exchange column (HiTrap SP column,Pharmacia) in Example 1. The lower the value is, the fewer the viablecells are.

FIG. 2 shows the cytotoxic activity (the absorbance at 450 nm) of eachfraction separated by a hydrophobic column (HiTrap Phenyl column,Pharmacia) in Example 1. The lower the value is, the fewer the viablecells are.

FIG. 3 shows the cytotoxic activity (the absorbance at 450 nm) of eachfraction separated by a gel filtration column (Pharmacia) in Example 1.The lower the value is, the fewer the viable cells are.

FIG. 4 shows the cytotocic activity (the absorbance at 450 nm) of eachfraction separated by a cation exchange column (Mono S column,Pharmacia) in Example 1. The lower the value is, the fewer the viablecells are.

FIG. 5 shows Native-PAGE profile of the purified apoptosis-inducingprotein from Helicobacter pylori.

FIG. 6 shows SDS-PAGE profile of the purified apoptosis-inducing proteinfrom Helicobacter pylori.

FIG. 7 shows the cytotoxic activity (the absorbance at 450 nm) of thepurified apoptosis-inducing protein from Helicobacter pylori examinedwith AGS cells and HeLa cells. The lower the value is, the fewer theviable cells are.

FIG. 8 shows the cytotoxic activity (the absorbance at 450 nm) of thepurified apoptosis-inducing protein from Helicobacter pylori examinedwith KATO III cells. The lower the value is, the fewer the viable cellsare.

FIG. 9 shows the rate (%) of apoptotic cells of AGS cells induced bypurified apoptosis-inducing protein from Helicobacter pylori.

FIG. 10 shows the amino acid sequence of the protein (567 amino acidresidues) transcribed from the HP1118 gene and the amino acid sequencesof subunit 1 (SEQ ID NO: 1) and subunit 2 (SEQ ID NO: 2) of theapoptosis-inducing protein from Helicobacter pylori.

FIG. 11 shows the schematic diagram of the apoptosis-inducing proteinfrom Helicobacter pylori.

DETAILED DESCRIPTION OF THE INVENTION

Since the protein complex of the present invention, composed by theeassociation of two kinds of proteins, has the apoptosis-inducingactivity of Helicobacter pylori, it can be used as the target moleculefor the treatment of gastritis and stomach ulcer induced by Helicobacterpylori.

For example, the protein complex can be prepared either by introducingthe genes encoding two subunits into expression vectors, e.g. in E.coli, separately and by mixing the subunits expressed independently, orby introducing a gene encoding both two subunits into an expressionvector, e.g. in E. coli and by expressing these subunits at the sametime.

After purification, the protein complex can be used to seek medicalagents inhibiting an apoptosis-inducing activity. The apoptosis-inducingactivity can be assayed by a conventional method for examining theapoptosis of gastric adenocarcinoma cell line.

The protein of the present invention can be applied to the developmentof a vaccine inhibiting the apoptosis-inducing activity of Helicobacterpylori.

For example, an animal could be immunized by an oral administration,transnasal administration or subcutaneous injection of the purified saidprotein complex as an antigen. Then, the animal is infected by thebacterium by an oral administration and is examined whether a defensiveeffect against the bacterium infection could be established. When thedefensive effect in the animal is positive, the effect could be examinedfor humans. Furthermore, if there are common antigens between saidprotein complex and substances produced by human cells, the amino acidsequence of the common antigen could be examined and removed from saidantigenic protein complex. A defensive effect against the infectioncould be examined by using the prepared protein, from which the commonantigenic part is removed. Finally, the protein thus prepared, with adefensive effect, could be used as a vaccine.

Moreover, a mutation could be introduced into the gene encoding theapoptosis-inducing protein of Helicobacter pylori and the proteinwithout the apoptosis-inducing activity, which is prepared from themodified gene, could be used as a vaccine.

Still furthermore, a novel Helicobacter pylori strain without thepathogenic activity could be prepared by disrupting the gene encodingthe apoptosis-inducing protein of Helicobacter pylori and could be usedas a vaccine.

Based on the nucleotide sequence of the gene encoding theapoptosis-inducing protein of Helicobacter pylori according to thepresent invention, it is possible to prepare PCR primers to amplify thegene. The PCR primers could be used to differentiate pathogenicHelicobacter pylori from nonpathogenic one.

The following examples illustrate this invention, however, it is notintended to limit the scope of this invention.

EXAMPLE 1

In this example, purification and identification of theapoptosis-inducing protein of Helicobacter pylori were conducted.

In this example, the apoptosis-inducing activity was measured as thecytotoxic activity in human gastric adenocarcinoma cell line AGS (ATCCCRL 1739) using Quick Cell Proliferation Assay Kit (Cat.BV-K301-10)(BioVision). The cell viability (surviving cell), from which anapoptosis-inducing activity is calculated, is based on the activity ofintracellular mitochondrial dehydrogenase activity as a marker enzymeand is measured by the absorbance at 450 nm after 1 hr incubation at 37°C. with the substrate of the enzyme. The absorbance indicates the cellviability. The more cytotoxic, the less the absorbance is according tothe mesurement.

First of all, Helicobacter pylori strain 26695 (ATCC CTL 700392) wasgrown in 1000 ml of BHI medium (Becton Dikkinson Co., Brain HeartInfusion N0. 237500) supplemented with 10% serum. After harvest bycentrifugation, the bacteria were suspended in PBS (Phosphate bufferedsaline, physiological saline (0.9% NaCl) adjusted to pH 7.4 with 50 mMsodium phosphate) and washed by centrifugation. The bacterial pellet wasresuspended in 50 ml of PBS and disrupted by a French press at 120 Mpa.After low speed centrifugation at 5000×g for 10 min to remove theunbroken bacteria, membrane fraction was sedimented by centrifuging at100,000×g for 2 hrs. The membrane fractions were suspended in 50 mMphosphate buffer (pH 6.0) containing 1 M NaCl and again centrifuged at100,000×g for 2 hrs. During the procedures, the apoptosis-inducingprotein was separated from membrane fractions and was solubilized in thesupernatant fraction.

The membrane fractions were suspended in 50 mM phosphate buffer (pH 6.0)containing 1 M NaCl and again centrifuged at 100,000×g for 2 hrs. Duringthe procedures, the apoptosis-inducing protein was separated frommembrane fractions and was solubilized in the supernatant fraction.

From the supernatant fraction, the apoptosis-inducing protein waspurified in the following way.

In the beginning, the supernatant fraction was dialyzed against 250 mMNaCl-containing buffer, loaded in a cation exchange column (HiTrap SPcolumn, Pharmacia), and fractionated by eluting with the gradient of250-500 mM NaCl. Then, each fraction was assessed on theapoptosis-inducing activity.

FIG. 1 shows the result. Fractions 10 to 12 contained the activity.These fractions were collected and used for the subsequent purificationsteps.

Then, the collected fraction containing the activity was loaded in ahydrophobic column (HiTrap Phenyl, Pharmacia) and fractionated byeluting with the gradient of 20-10% ammonium sulfate. Then, eachfraction was assessed on the apoptosis-inducing activity. FIG. 2 showsthe results. Fractions 8 to 12 contained the activity. These fractionswere collected and used for the following purification steps.

Then, the collected fraction containing the activity was loaded in agel-filtration column (Pharmacia), fractionated by 50 mM phosphatebuffer (pH 6.0) containing 500 mM NaCl and each fraction was assessed onthe apoptosis-inducing activity. FIG. 3 shows the results. Fractions 12to 14 contained the activity. These fractions were collected and usedfor the following purification steps.

Then, the collected fraction containing the activity was dialyzedagainst 250 mM NaCl-containing buffer, loaded in a cation exchangecolumn (MonoS column, Pharmacia) and fractionated by eluting with thegradient of 250-500 mM NaCl. Then, each fraction was assessed on thecytotoxic activity. FIG. 4 shows the results. Fractions 7 and 8contained the activity. These fractions were collected.

The purified protein obtained by the above procedures was analyzed by anative PAGE under acid condition. As shown in FIG. 5, a single band wasobserved. The single band was subsequently analyzed by 2D SDS-PAGE. Asshown in FIG. 6, the band was separated into two bands (their molecularweight were about 40 kDa and 22 kDa).

In native gel, which is free from strong detergent such as SDS in acrylamide, the three dimensional structure of the protein is preserved.Observation of the single band indicates that the protein is composed ofa single complex in its native state.

On the other hand, in SDS-PAGE, the three dimensional structure of theprotein is disrupted by SDS and multiple protein subunits are separated.The result that a single band in native PAGE is separated into two bandsin SDS-PAGE indicates that the protein complex is composed of the twoprotein subunits.

In other words, the apoptosis-inducing protein from Helicobacter pyloriis a protein constructed by an association of two kinds of subunits.

EXAMPLE 2

In this example, the apoptosis-inducing activity of the protein purifiedin example 1 was examined in human gastric adenocarcinoma cell line AGS(ATCC CRL 1739), in human gastric carcinoma cell line KATO III (HumanScience Research Resources Bank, Cat No. JCRB0611 (ATCC, HTB-103)) andin human uterine cervix cancer-derived HeLa cell (Human Science ResearchResources Bank, Cat No. JCRB9004).

The cytotoxic effect was assayed using WST-1 kit (Roche Diagnotics). Theresults are shown in FIGS. 7 and 8.

As shown in these figures, the protein purified in example 1 has thecytotoxic activity in all the above cell lines.

Also, the induction of apoptosis in AGS cells by the protein wasexamined: Trypsinized apoptotic and control cells were collected andstained with a fluorescent dye (Hoechst 33342), which stains cellularnuclei. Since the nuclei of apoptotic cells are highly condensed andfragmented, apoptotic cells are easily differentiated from control cellsunder fluorescence microscopy. Cells were put on a slide glass, thenumber of apoptotic cells and control cells were counted and thepercentage of apoptotic cells in total cells was enumerated. At leasttotal 500 cells were counted for each experiment.

The results are shown in FIG. 9. It was confirmed that the cytotoxiceffect of the protein against AGS cells was mainly ascribed toapoptosis.

EXAMPLE 3

The amino acid sequencing of N-terminal end of the two subunits purifiedin example 1 indicated that both subunits were encoded by the HP1118gene. FIG. 10 shows the amino acid sequence of two subunits (subunits 1and 2) together with that of the HP1118 gene. More specifically, thesubunit 1 (SEQ ID NO: 1) and the subunit 2 (SEQ ID NO: 2) comprise aminoacids 27-379 and 380-567, respectively, of the amino acid sequenceencoded by the HP1118 gene.

As above described, the apoptosis-inducing protein of Helicobacterpylori is a protein complex composed of two protein subunits as shown inFIG. 11: one (subunit 1) is produced by the cleavage between 26^(th) and27^(th) and between 379^(th) and 380^(th) of the amino acid residues ofthe protein transcribed from the HP1118 gene and the other (subunit 2)is produced by the cleavage between 379^(th) and 380^(th) of the aminoacid residues of said protein.

1. An apoptosis-inducing protein of Helicobacter pylori comprising twokinds of proteins having an amino acid sequence of SEQ ID NO: 1 or 2, ortwo kinds of proteins having an amino acid sequence of SEQ ID NO: 1 or 2wherein one or several amino acids with respect to the amino acidsequence of SEQ ID NO: 1 or 2 is/are deleted, substituted or added, andhaving an apoptosis-inducing activity in human gastric adenocarcinomacell line.
 2. A protein comprising either of the following proteins: (a)a protein having an amino acid sequence of SEQ ID NO: 1, or (b) aprotein having an amino acid sequence comprising a deletion,substitution or addition of one or several amino acids with respect tothe amino acid sequence of SEQ ID NO: 1, and having anapoptosis-inducing activity in human gastric adenocarcinoma cell line byassociation with the protein comprising the amino acid sequence of SEQID NO:
 2. 3. A protein comprising either of the following proteins: (a)a protein having an amino acid sequence of SEQ ID NO: 2, or (b) aprotein having an amino acid sequence comprising a deletion,substitution or addition of one or several amino acids with respect tothe amino acid sequence of SEQ ID NO: 2, and having anapoptosis-inducing activity in human gastric adenocarcinoma cell line byassociation with the protein comprising the amino acid sequence of SEQID NO:
 1. 4. The apoptosis-inducing protein of Helicobacter pyloricomprising: (1) a protein comprising either of the following proteins:(a) a protein having an amino acid sequence of SEQ ID NO: 1, or (b) aprotein having an amino acid sequence comprising a deletion,substitution or addition of one or several amino acids with respect tothe amino acid sequence of SEQ ID NO: 1, and having anapoptosis-inducing activity in human gastric adenocarcinoma cell line byassociation with the protein comprising the amino acid sequence of SEQID NO: 2; and (2) a protein comprising either of the following proteins:(a) a protein having an amino acid sequence of SEQ ID NO: 2, or (b) aprotein having an amino acid sequence comprising a deletion,substitution or addition of one or several amino acids with respect tothe amino acid sequence of SEQ ID NO:
 2. and having anapoptosis-inducing activity in human gastric adenocarcinoma cell line byassociation with the protein comprising the amino acid sequence of SEQID NO:
 1. 5. A vaccine comprising the protein of claim 1, wherein a partof the amino acid sequence of SEQ ID NO: 1 and 2 is deleted, substitutedor added, and said protein has little or no apoptosis-inducing activityin gastric adenocarcinoma cell line and possesses a defensive effectagainst the infection of Helicobacter pylori.
 6. A gene encoding theprotein of claim
 2. 7. A gene encoding the protein of claim
 3. 8. Amethod for distinguishing between pathogenic strains and nonpathogenicstrains of Helicobacter pylori, comprising a step of amplifying at leastone of the whole or a part of: (1) a gene encoding a Protein comprisingeither of the following Proteins: (a) a protein having an amino acidsequence of SEQ ID NO: 1, or (b) a protein having an amino acid sequencecomprising a deletion, substitution or addition of one or several aminoacids with respect to the amino acid sequence of SEQ ID NO: 1, andhaving an apoptosis-inducing activity in human gastric adenocarcinomacell line by association with the protein comprising the amino acidsequence of SEQ ID NO: 2; and (2) a gene encoding a Protein comprisingeither of the following Proteins: (a) a protein having an amino acidsequence of SEQ ID NO: 2, or (b) a protein having an amino acid sequencecomprising a deletion, substitution or addition of one or several aminoacids with respect to the amino acid sequence of SEQ ID NO: 2, andhaving an apoptosis-inducing activity in human gastric adenocarcinomacell line by association with the protein comprising the amino acidsequence of SEQ ID NO:
 1. 9. A vaccine comprising: (a) a protein havingan amino acid sequence of SEQ ID NO: 1, or (b) a protein having an aminoacid sequence comprising a deletion, substitution or addition or one orseveral amino acids with respect to the amino acid sequence of SEQ IDNO: 1, and having an apoptosis-inducing activity in human gastricadenocarcinoma cell line by association with the protein comprising theamino acid sequence of SEQ ID NO: 2; and (c) a protein having an aminoacid sequence of SEQ ID NO: 2, or (d) a protein having an amino acidsequence comprising a deletion, substitution or addition of one orseveral amino acids with respect to the amino acid sequence of SEQ IDNO: 2, and having an apoptosis-inducing activity in human gastricadenocarcinoma cell line by association with the protein comprising theamino acid sequence of SEQ ID NO: 1; wherein a part of the amino acidsequence of SEQ ID NO: 1 and 2 is deleted, substituted or added, andsaid vaccine has little or no apoptosis-inducing activity in gastricadenocarcinoma cell line and possesses a defensive effect against theinfection of Helicobacter pylori.
 10. A gene encoding: (a) a proteinhaving an amino acid sequence of SEQ ID NO: 1, or (b) a protein havingan amino acid sequence comprising a deletion, substitution or additionof one or several amino acids with respect to the amino acid sequence ofSEQ ID NO: 1, and having an apoptosis-inducing activity in human gastricadenocarcinoma cell line by association with the protein comprising theamino acid sequence of SEQ ID NO:
 2. 11. A gene encoding: (c) a proteinhaving an amino acid sequence of SEQ ID NO: 2, or (d) a protein havingan amino acid sequence comprising a deletion, substitution or additionof one or several amino acids with respect to the amino acid sequence ofSEQ ID NO: 2, and having an apoptosis-inducing activity in human gastricadenocarcinoma cell line by association with the protein comprising theamino acid sequence of SEQ ID NO:
 1. 12. A method for distinguishingbetween pathogenic strains and nonpathogenic strains of Helicobacterpylori, comprising the step of amplifying at least one of the whole or apart of the gene encoding: (a) a protein having an amino acid sequenceof SEQ ID NO: 1, or (b) a protein having an amino acid sequencecomprising a deletion, substitution or addition of one or several aminoacids with respect to the amino acid sequence of SEQ ID NO: 1, andhaving an apoptosis-inducing activity in human gastric adenocarcinomacell line by association with the protein comprising the amino acidsequence of SEQ ID NO: 2; and the whole or a part of the gene encoding:(c) a protein having an amino acid sequence of SEQ ID NO: 2, or (d) aprotein having an amino acid sequence comprising a deletion,substitution or addition of one or several amino acids with respect tothe amino acid sequence of SEQ ID NO: 2, and having anapoptosis-inducing activity in human gastric adenocarcinoma cell line byassociation with the protein comprising the amino acid sequence of SEQID NO:
 1. 13. A method for suppressing the apoptosis-inducing activityof Helicobacter pylori, comprising the step of inhibiting the expressionof the apoptosis-inducing protein of Helicobacter pylori in gastricadenocarcinoma cell line, which comprises two kinds of proteins havingan amino acid sequence of SEQ. ID NO: 1 or 2, or two kinds of proteinshaving an amino acid sequence of SEQ ID NO: 1 or 2 wherein one orseveral amino acids with respect to the amino acid sequence of SEQ IDNO: 1 or 2 is/are deleted, substituted or added, and having anapoptosis-inducing activity in human gastric adenocarcinoma cell line.14. A method for producing a vaccine to suppress the apoptosis-inducingactivity by Helicobacter pylori, comprising the steps of immunizingnon-human animals with the apoptosis-inducing proteins of Helicobacterpylori as an antigen, which comprises two kinds of proteins having anamino acid sequence of SEQ ID NO: 1 or 2, or two kinds of proteinshaving an amino acid sequence of SEQ ID NO: 1 or 2 wherein one orseveral amino acids with respect to the amino acid sequence of SEQ IDNO: 1 or 2 is/are deleted, substituted or added, and having anapoptosis-inducing activity in human gastric adencarcinoma cell line,and selecting the proteins with the defensive effect against theinfection by the bacterium on the animal administered the bacterium. 15.A method of claim 14, which comprises selecting the proteins with thedefensive effect against the infection on human out of the proteins withthe defensive effect against the infection on the animal model.
 16. Avaccine to suppress the apoptosis-inducing activity of Helicobacterpylori, comprising the protein, which is obtained by introduction of amutation in a gene encoding an apoptosis-inducing protein ofHelicobacter pylori comprising two kinds of proteins having an aminoacid sequence of SEQ ID NO: 1 or 2, or two kinds of proteins having anamino acid sequence of SEQ ID NO: 1 or 2 wherein one or several aminoacids with respect to the amino acid sequence of SEQ ID NO: 1 or 2is/are deleted, substituted or added, and having an apoptosis-inducingactivity in human gastric adenocarcinoma cell line.
 17. A vaccine tosuppress the apoptosis-inducing activity of Helicobacter pylori,comprising non-pathogenic Helicobacter pylori, which is obtained bydisruption of a gene encoding an apoptosis-inducing protein ofHelicobacter pylori comprising two kinds of proteins having an aminoacid sequence of SEQ ID NO: 1 or 2, or two kinds of proteins having anamino acid sequence of SEQ ID NO: 1 or 2 wherein one or several aminoacids with respect to the amino acid sequence of SEQ ID NO: 1 or 2is/are deleted, substituted or added, and having an apoptosis-inducingactivity in human gastric adenocarcinoma cell line.
 18. Use of anapoptosis-inducing protein of Helicobacter pylori, as a vaccine tosuppress the apoptosis-inducing activity of Helicobacter pylori, whichcomprises two kinds of proteins having an amino acid sequence of SEQ IDNO: 1 or 2, or two kinds of proteins having an amino acid sequence ofSEQ ID NO: 1 or 2 wherein one or several amino acids with respect to theamino acid sequence of SEQ ID NO: 1 or 2 is/are deleted, substituted oradded, and having an apoptosis-inducing activity in human gastricadenocarcinoma cell line, and selecting the proteins with the defensiveeffect against the infection by the bacterium on the animal administeredthe bacterium.